Quinolonecarboxylic acid derivative

ABSTRACT

(−)-7-[(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R, 2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid.1HCl.1H 2 O and antibacterial compositions containing this compound. The compound of the present invention exhibits not only excellent antibacterial activity and safety, but also remarkable stability against light and humidity, and is thus useful as an antibacterial agent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to optically active quinolonecarboxylicacid derivatives which exhibit high antibacterial activity and safetyand have remarkable stability, and to an antibacterial compositioncontaining such a derivative.

2. Related Art

Quinolonecarboxylic acid derivatives are a class known as syntheticantibacterial agents. Particularly, compounds of the following formula(I) having a 1,2-cis-2-halogenocyclopropyl group on the nitrogen atom atthe 1-position of the quinolone skeletone exhibit strong antibacterialactivity and safety, and thus are known to be useful as pharmaceuticalagents (Japanese patent No. 2714597 and 2917010);

wherein R¹ is an amino group, a methylamino group, a hydroxyl group, athiol group, or a hydrogen atom; and R² is a substituent selected fromthe group consisting of the following groups:

(wherein R³, R⁴, R⁵, and R⁶ each independently represents a hydrogenatom or a C1-C6 alkyl group, R¹⁰ and R¹¹ each independently represents ahydrogen atom or a C1-C6 alkyl group, R¹² and R¹³ each independentlyrepresents a hydrogen atom or a C1-C6 alkyl group or R¹² and R¹³ mayform a polymethylene chain of from 2 to 5 carbon atoms), or a3-hydroxypyrrolidinyl group which may have a C1-C6 alkyl group; Arepresents C-X³ or a nitrogen atom; X¹ and X² each independentlyrepresents a halogen atom; X³ represents a halogen atom, a C1-C6 alkylgroup, a C1-C6 alkoxy group, trifluoromethyl group, or a hydrogen atom;and Z represents a hydrogen atom, a C1-C6 alkyl group, a C1-C6alkoxyalkyl group, a phenylalkyl group of C1-C6 alkyl, a phenyl group,an acetoxymethyl group, a pivaloyloxymethyl group, an ethoxycarbonyloxygroup, a chorine group, a dimethylaminoethyl group, a 5-indanyl group, aphthalidinyl group, a 5-substituted-2-oxo-1,3-dioxol-4-ylmethyl group,or a 3-acetoxy-2-oxobutyl group; with the case in which R² is a3-aminopyrrolidinyl group and R¹ and X³ are hydrogen atoms beingexcluded.

A variety of fluoroquinolone synthetic antibacterial agents have beendeveloped to provide clinical drugs for chemotherapy which are effectivein the treatment of a broad range of systemic infectious diseases.Nevertheless, demands still exist for compounds which exhibit higherantibacterial activity, which are safer than the previous compounds, andin addition, which are endowed with excellent stability against lightand humidity.

SUMMARY OF THE INVENTION

In view of the foregoing, the present inventors, focusing onN₁-(1,2-cis-2-fluorocyclopropyl)-substituted pyrrolidonecarboxylic acidsdisclosed in the aforementioned Japanese Patent No. 2714597, haveperformed further research and have found that amonohydrochloride•monohydrate of compound No. 41 disclosed in JapanesePatent No. 2714597—but the disclosure being limited only to the chemicalformula of its free form, particularly,(−)-7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (hereinafter referred to as compound (1a); compound (1a)corresponds to a free form and is represented by the following formula(1a))—exhibits not only excellent antibacterial activity and safety, butalso exceptionally excellent stability against light and humidity ascompared with other acid-adduct salts, and is thus useful as anantibacterial agent. The present invention has been accomplished on thebasis of these findings.

Accordingly, the present invention provides a compound represented bythe following formula (1):

which is(−)-7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid monohydrochloride monohydrate (hereinafter referred to as compound(1); compound (1) is a monohydrochloride-monohydrate of compound (1a)),and antibacterial agents containing the compound (1).

The present invention also provides an antibacterial agent containing acompound represented by the following formula (1a), an acid-adduct saltthereof, or a hydrate of the following formula (1a) compound or theacid-adduct salt.

The present invention further provides use of the above-describedcompound of formula (1a), an acid-addition salt thereof, or a hydrate ofthe formula (1a) compound or the acid-addition salt in the manufactureof a drug for the treatment of infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a powder X-ray diffraction spectrum of compound (1).

FIG. 2 shows an infrared absorption spectrum of compound (1).

FIG. 3 is a graph showing the weight change of compound (1) under 5-95%RH.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Compound (1a) can be produced from a compound (2) at high yield inaccordance with the following reaction scheme. Briefly, an aminecompound (3) is reacted with compound (2) (note: both of these compoundscan be obtained through the method described in Japanese Patent No.2714597), and treatment of the resultant compound (1b) with a proticsolvent yields compound (1a). Accordingly, compound (1b) is a usefulsynthetic intermediate in the production of compound (1a).

With regard to the reaction conditions under which compound (1b) isproduced from compound (2), for example, a solution of dihydrochlorideof amine (3) and triethylamine in dimethylsulfoxide is stirred(ordinary, for at most 2 to 3 hours is enough) at room temperature asolution of, and thereafter, compound (2) is added thereto and arereacted at room temperature for 10 minutes to several hours. In thisreaction, instead of the dihydrochloride of amine (3), a correspondingfree base (3) or a salt of any other type may be used. The salt of anyother type may be, for example, monohydrochloride, or a mono- ordi-salt—here, “mono-” and “di-” are both with respect to amine (3)—oforganic or inorganic acid (other than HCl). Alternatively, the salt maybe in the form of a hydrate or a solvate. Examples of the organic orinorganic acid suitable for acid-addudct salt(other than HCl) includesulfuric acid, nitric acid, hydrofluoric acid, hydrobromic acid,hydroiodic acid, p-toluenesulfonic acid, methanesulfonic acid,trifluoroacetic acid, trichloroacetic acid, acetic acid, formic acid,maleic acid, and fumaric acid. Examples of the reaction solvent inaddition to dimethylsulfoxide include N,N-dimethylformamide,N,N-dimethylacetamide, and N-methylpyrrolidone. In the above-mentionedexample in which triethylamine is used, when a free base of amine (3) isemployed, the amount of triethylamine is preferably one equivalent ormore, more preferably two equivalents or more. When a salt of amine (3)is employed, the amount of triethylamine is preferably equal to or morethan a total of the equivalent required for rendering the salt into afree base and the equivalent required for capturing hydrogen fluoridegenerated from the reaction. Instead of triethylamine, any other organicor inorganic bases, such as 4-(dimethylamino)pyridine or potassiumcarbonate, may be used.

The production step of compound (1a) from compound (1b) may be performedby, for example, dissolving compound (1b) in aqueous ethanol, addingtriethylamine to the resultant mixture, followed by refluxing for a fewhours. The aqueous ethanol may be replaced by another protic solvent,such as aqueous isopropanol. The protic solvents miscible with water aresuitable for this process, and these which can at least dissolvecompound (1b) when heated is preferable. Note that triethylamine is notnecessarily added.

Compound (1a) may also be produced from a compound (2) at high yield inaccordance with the following reaction scheme. Briefly, amine compound(5) is reacted with compound (2), to thereby obtain a carboxyliccompound (7), and thereafter the protective group for the amino group isremoved.

(wherein R represents an alkoxycarbonyl group, an aralkyloxycarbonylgroup, an acyl group, an alkyl group, an aralkyl group, an alkoxyalkylgroup (all of these may have substituent(s)), or a substituted silylgroup). Accordingly, compounds (6) and (7) are useful as syntheticintermediates in the production of compound (1a).

In compound (6), R serves as a protective group for an amino group, andis an alkoxycarbonyl group which may have substituent(s), anaralkyloxycarbonyl group which may have substituent(s), an acyl groupwhich may have substituent(s), an alkyl group which may havesubstituent(s), an aralkyl group which may have substituent(s), analkoxyalkyl group which may have substituent(s), or a substituted silylgroup. Of these species, R is preferably alkoxycarbonyl group, anaralkyloxycarbonyl group, an acyl group, or a silyl group, withalkoxycarbonyl and aralkyloxycarbonyl being more preferred. Specificexamples of the alkoxycarbonyl group which may have substituent(s)include a tert-butoxycarbonyl group (Boc) and a2,2,2-trichloroethoxycarbonyl group, wherein the tert-butoxycarbonylgroup is preferred. Specific examples of the aralkyloxycarbonyl whichmay have substituent(s) group include a benzyloxycarbonyl group, ap-methoxybenzyloxycarbonyl group, and a p-nitrobenzyloxycarbonyl group,wherein the p-methoxybenzyloxycarbonyl group and thep-nitrobenzyloxycarbonyl group are preferred. Specific examples of theacyl group which may have substituent(s) include an acetyl group, amethoxyacetyl group, a trifluoroacetyl group, a chloroacetyl group, apyvaloyl group, a formyl group, and a benzoyl group. Of these species, atrifluoroacetyl group, a chloroacetyl group, a pyvaloyl group, and aformyl group are preferred. Specific examples of the alkyl group whichmay have substituent(s) include a tert-butyl group. Examples of thearalkyl group which may have substituent(s) include a benzyl group, ap-nitrobenzyl group, p-methoxybenzyl group, and a triphenylmethyl group,with the p-methoxybenzyl group and triphenylmethyl group beingpreferred. Examples of the alkoxyalkyl group which may havesubstituent(s) include a methoxymethyl group, a tert-butoxymethyl group,a 2,2,2-trichloroethoxymethyl group, and a tetrahydrofuranyl group, withthe tert-butoxymethyl group and tetrahydrofuranyl group being preferred.Examples of the substituted silyl group include a trimethylsilyl group,an isopropyldimethylsilyl group, a tert-butyldimethylsilyl group, atribenzylsilyl group, and a tert-butyldiphenylsilyl group, with theisopropyldimethylsilyl group, a tert-butyldimethylsilyl group beingpreferred.

However, R is not limited only to the above-listed species, and anygroup which is ordinarily used for the protection of an amino group issuitable for the present invention and the protective group selectedfrom the group consisting of alkoxycarbonyl groups, aralkyloxycarbonylgroups, acyl groups, alkyl groups, aralkyl groups, alkoxyalkyl groups,and silyl groups may serve as R.

With regard to the reaction conditions under which compound (6) isproduced, for example, an amine (5) may be used to cause a reaction indimethylsulfoxide in the presence of trimethylamine at room temperaturefor several hours to one day. Amine (5) may be in the form of a freebase, or a salt of an organic or inorganic acid. Preferably, amine (5)is used in an mount of one equivalent or more. When a free base of amine(5) is used, a required amount of triethylamine is one or moreequivalents, more preferably two or more equivalents. Alternatively,when a salt of amine (5) is employed, the amount of triethylamine ispreferably equal to or more than a total of the equivalent required forrendering the salt into a free base, and further equivalent required forcapturing hydrogen fluoride generated from the reaction. Examples ofsalts of amine (5) include salts of organic or inorganic acid, such ashydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid,hydrobromic acid, hydroiodic acid, p-toluenesulfonic acid,methanesulfonic acid, trifluoroacetic acid, trichloroacetic acid, aceticacid, formic acid, maleic acid, and fumaric acid. These salts may be ina form of hydrates or solvates. Examples of the reaction solvent inaddition to dimethylsulfoxide include N,N-dimethylformamide,N,N-dimethylacetamide, and N-methylpyrrolidone. In the above-mentionedexample in which triethylamine is used, the triethylamine may bereplaced by any one of organic or inorganic bases, such as4-(dimethylamino)pyridine or potassium carbonate.

Conversion of compound (6) to compound (7) may be performed by, forexample, dissolving compound (6) in aqueous ethanol, addingtriethylamine to the resultant mixture, followed by refluxing for a fewhours. The aqueous ethanol may be replaced by another protonic solvent,such as aqueous isopropanol. The protic solvents miscible with water aresuitable for this process, and these which can at least dissolvecompound (1b) when heated is preferable. Note that triethylamine is notnecessarily added.

In the step of producing compound (1a) from compound (7), the conditionsunder which the protective group R is removed must conform with theproperties of the protective group R. Typical exemplary cases are asfollows. When R is a tertbutoxycarbonyl group (Boc), deprotection may becarried out through treatment with an organic or inorganic acid such ashydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid,hydrobromic acid, hydroiodic acid, p-toluenesulfonic acid,methanesulfonic acid, trifluoroacetic acid, trichloroacetic acid, aceticacid, or formic acid. The temperature at which deprotection is carriedout is suitably chosen from the range of −30 to 100° C., in accordancewith the type and concentration of the acid employed and with theproperty of the solvent. Similarly, deprotection may be carried out fora p-methoxybenzyloxycarbonyl group, an acetyl group, a pyvaloyl group, amethoxyacetyl group, a formyl group, a tert-butyl group, a methoxymethylgroup, a tert-butoxymethyl group, a tetrahydrofuranyl group, atrimethylsilyl group, a triphenylmethyl group, or from similar groupsthrough treatment with an acid that is suitably chosen from amonghydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid,hydrobromic acid, hydroiodic acid, p-toluenesulfonic acid,methanesulfonic acid, trifluoroacetic acid, and trichloroacetic acid. A2,2,2-trichloroethoxycarbonyl group and a 2,2,2-trichloroethoxymethylgroup may be removed by using a combination of zinc and an acid (HCl oracetic acid). Deprotection of a benzyloxycarbonyl group, ap-methoxybenzyloxycarbonyl group, a p-nitrobenzyloxycarbonyl group, abenzyl group, a p-nitrobenzyl group, a p-methoxybenzyl group, or atriphenylmethyl group can be carried out through catalytic reduction.Acyl groups such as an acetyl group, a methoxyacetyl group, atrifluoroacetyl group, a chloroacetyl group, a pyvaloyl group, a formylgroup, and a benzoyl group may be removed through treatment with an acidsuch as HCl or an alkali such as NaOH. Silyl groups such as atrimethylsilyl group, an isopropyldimethylsilyl group, atert-butyldimethylsilyl group, a tribenzylsilyl group, and atert-butyldiphenylsilyl group may be removed by use of an acid orfluoride ions. In this case, example of the acid include acetic acid,hydrochloric acid, and hydrofluoric acid, and a suitable acid must bechosen so as to be in conformity with the properties of the silyl group.As for the source of the fluoride ions, tetrabutylammonium fluoride maybe used. A chloroacetyl group can be removed by use of thiourea. Detailsof the conditions under which deprotection is carried out are ordinaryones and not particularly limited.

With reference to compound (6), when R is a tert-butoxycarbonyl group ora similar group which can be removed by an acid, compound (1a) can beobtained through direct treatment of compound (6) with an organic orinorganic acid such as hydrochloric acid, sulfuric acid, nitric acid,hydrofluoric acid, hydrobromic acid, hydroiodic acid, p-toluenesulfonicacid, methanesulfonic acid, trifluoroacetic acid, or trichloroaceticacid.

In the above-described two production methods, compound (1a) may beobtained in a free form or as a salt. Examples of the salt include asalt of an inorganic or organic acid, such as hydrochloric acid,sulfuric acid, nitric acid, hydrofluoric acid, hydrobromic acid,hydroiodic acid, p-toluenesulfonic acid, methanesulfonic acid,trifluoroacetic acid, trichloroacetic acid, acetic acid, maleic acid, orfumaric acid; and a salt of an alkali or alkaline earth metal such assodium, potassium, calcium, or lithium. In either case of compound (1a)being in its free form or a salt, the compound (1a) may be obtained inthe form of a solvate. Examples of the solvate include those formed withwater, ethanol, propanol, acetonitrole, or acetone; and hydrates whichmay be formed through absorption of moisture in air.

The compound (2) which is used in the aforementioned two reactionschemes for producing compound (1a) may be prepared through thefollowing reaction path.

In the above scheme, in order to produce compound (2) from compound(12), an ether complex of boron trifluoride may be used. Alternatively,compound (2) may be obtained without obtaining compound (12) but bytreating compound (11) with tetrafluoroboric acid as shown below.

Conversion from compound (1a) to compound (1) can be carried out by, forexample, suspending compound (1a) in an alcoholic solvent such as2-propanol or ethanol, dissolving the suspension by the addition of HCl,and subsequently crystallizing from an alcoholic solvent such as2-propanol or ethanol.

The thus-obtained compounds (1a), their acid-addition salts, or salts ofthe compounds (1a), in particular compounds (1), which aremonohydrochlorides.monohydrates of compounds (1a), exhibit moreexcellent antibacterial activity and higher stability against light andhumidity than, let alone other compounds, compound Nos. 9a, 9b, 13b,18a, 18b, 26bb, 26aa, 26ba, 26ab, 31a, 31b, 34b, 54b, 56b, 52bb, or 85bbdisclosed in Japanese Patent No. 2714597 or 2917010, and are thus usefulas antibacterial agents. Preferred acid-addition salts of compounds (1a)applicable for antibacterial agents are hydrochloric acid salts. Amongthe mentioned compounds (1a), their acid-addition salts, and salts ofthe compounds (1a) or the acid-addition salts, compounds (1) areparticularly preferred.

More preferred compounds (1); i.e., monohydrochlorides.monohydrates ofcompounds (1a), are those exhibiting characteristic peaks in thevicinity of angles of diffraction (2θ) of 6.9, 10.5, 14.4, 23.1, 26.9,and 27.8(°) when subjected to powder X-ray diffractometry (see FIG. 1).The compounds (1) satisfying the above conditions do not absorb ordesorb moisture under humidity conditions of 5 to 95% RH, and thus hasexcellent hygroscopic stability.

The compounds of the present invention, exhibiting strong antibacterialactivity and excellent stability against light and humidity, are usefulas pharmaceuticals for the treatment of humans, animals, and fish.

When the compounds of the present invention are used as pharmaceuticalsfor humans, the daily dose for an adult is from 50 mg to 1 g, preferablyfrom 100 mg to 300 mg. When the compounds are administered to animals,the dose may vary depending on the purpose of administration(therapeutic or preventive), the species and size of the animal to betreated, the nature of the pathogen that infected the animal, and theseverity of the pathological condition. However, the daily dose istypically from 1 mg to 200 mg, preferably from 5 mg to 100 mg, per kg ofthe body weight of the animal. This dose is administered once a day or2-4 divided times a day. The daily dose may exceed the above-mentionedranges.

Antibacterial compositions containing the aforementioned compounds ofthe present invention can be formulated in a variety of formulationsthrough any ordinary methods in accordance with the manner of theadministration. Examples of the peroral form of the antibacterialformulations containing the compound of the present invention as anactive ingredient include tablets, powders, granules, capsules,solutions, syrups, elixirs, and oil-based or water-based suspensions.

Injections may contain a stabilizer, a preservative, or a solubilizingagent. Therefore, a solution which may contain any of these auxiliaryagents may be placed in a container, and a solid preparation may beformulated through, for example, freeze-drying, thereby yielding apharmaceutical product for bed-side preparation. Moreover, one dose or aplurality of doses may be contained in a single container.

When the composition of the present invention is formulated intoexternal agents, examples of the external agents include solutions,suspensions, emulsions, ointments, gels, creams, lotions, and sprays.

Solid preparations may contain, in addition to the compound of thepresent invention, pharmaceutically acceptable additives includingfillers, volume-increasing agents, binders, disintegrants, dissolutionaccelerators, wetting agents, and lublicants, as needed.

Examples of liquid preparations include solutions, suspensions, andemulsions. Liquid preparations optionally contain suspension aids andemulsifiers as additives therefor.

EXAMPLES

The present invention will be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Referential Example 1

Ethyl 3-dimethylamino-2-(3-methoxy-2,4,5-trifluorobenzoyl) acrylate:

Thionyl chloride (109.4 ml; 1500 mmol) was added dropwise at roomtemperature to a suspension containing 3-methoxy 2,4,5-trifluorobenzoicacid (206.1 g; 1000 mmol), N,N-dimethylformamide (2 ml), and toluene(2000 ml). After completion of the addition, the reaction mixture wasstirred in an oil bath at 80° C. for 16 hours. The reaction mixture wascooled, and the cooled solution was concentrated under reduced pressure.Toluene was added to the residue, and the mixture was concentratedagain. The procedure from the toluene addition to the concentration wasrepeated two more times, to thereby yield an acid chloride.

Ethyl 3-dimethylamino acrylate (171.8 g; 1200 mmol) and triethylamine(184.0 ml; 1320 mmol) were added to dry tetrahydrofuran (1500 ml). Tothe solution, a solution of the above-prepared acid chloride in drytetrahydrofuran (500 ml) was added dropwise under ice cooling. Aftercompletion of the addition, the reaction suspension was refluxed for 5hours, followed by cooling. The cooled reaction mixture was concentratedunder reduced pressure, and water (1500 ml) and dichloromethane (1500ml) were added to the residue, followed by stirring. The dichloromethanelayer was collected, and the aqueous layer was extracted withdichloromethane (1000 ml). The combined dichloromethane layer was washedwith saturated brine (1500 ml), and dried over anhydrous sodium sulfate,followed by filtration. The filtrate was concentrated under reducedpressure, and the residue was subjected to silica gel columnchromatography. Fractions eluted with n-hexane/ethyl acetate (1:1) wereconcentrated, followed by drying under reduced pressure, to therebyyield 270.3 g of the title compound as yellowish white creamy matter(yield: 81.6%).

¹H-NMR(400 MHz, CDCl₃)δ: 1.02(3H, t, J=7.08 Hz), 2.88(3H, br), 3.32(3H,br), 4.00(2H, q, J=7.08 Hz), 7.09-7.13(1H, m), 7.83(1H, s).

Referential Example 2

Ethyl 3-[(1R,2S)-2-fluoro-1-cyclopropylamino]-2-(3-methoxy-2,4,5-trifluorobenzoyl)acrylate:

Ethyl 3-dimethylamino-2-(3-methoxy-2,4,5-trifluorobenzoyl) acrylate(260.5 g; 786.3 mmol) was dissolved in dichloromethane (2200 ml), and(1R, 2S)-2-fluoro-1-cyclopropylamine p-toluene sulfonate (223.6 g; 904.2mmol) was added to the solution. The resultant suspension was cooled to−15° C., and a solution of triethylamine (138.6 ml; 994.6 mmol) indichloromethane (300 ml) was added dropwise to the suspension over 40minutes while being stirred. After completion of the addition, thetemperature was maintained for 1 hour, and then the suspension wascooled on ice for 1 hour, followed by stirring at room temperature for14 hours. Dichloromethane (1000 ml) and water (2000 ml) were added tothe reaction mixture, and the dichloromethane layer was collected. Theaqueous layer was extracted with dichloromethane (500 ml), and thecombined organic layer was washed with saturated brine (1000 ml),followed by drying over anhydrous sodium sulfate. The mixture wasfiltered, and the filtrate was concentrated under reduced pressure, tothereby yield 227.5 g of the title compound as yellow creamy matter(yield: 97.7%). The reaction product was a mixture of geometricalisomers (E and Z isomers). The product was used for the subsequentreaction without further purification.

¹H-NMR(400 MHz, CDCl₃)δ: 0.97, 1.09(total 3H, each t, J=7.08 Hz),1.21-1.37(2H, m), 2.90-2.99(1H, m), 4.01(3H, s), 4.03, 4.06(total 2H,each q, J=7.08 Hz), 4.73(1H, dm, J=63.72 Hz), 6.86-6.92, 6.98-7.04(total 1H, each m), 8.16, 8.23(total 1H, each d, J=13.67 Hz)

Referential Example 3

Ethyl 6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylate:

The above-synthesized crude ethyl 3-[(1R,2S)-2-fluoro-1-cyclopropylamino]-2-(3-methoxy-2,4,5-trifluorobenzoyl)acrylate (276.2 g; 764.5 mmol) was dissolved in dryN,N-dimethylformamide (2000 ml), and potassium carbonate (317.0 g; 2.293mmol) was suspended in the solution under ice cooling, followed bystirring for 72 hours at room temperature. Hydrochloric acid (2N) wasadded dropwise slowly to the reaction mixture so as to adjust the pH ofthe resultant suspension to about 3 while the suspension was stirredunder ice cooling. The suspension was stirred for 30 minutes at roomtemperature, and crystals precipitated were collected by filtration. Thethus-obtained crystals were sequentially washed with an excessive amountof purified water, a small amount of cold ethanol, and an excessiveamount of diethylether, followed by drying under reduced pressure at 70°C., to thereby yield 213.4 g of the title compound as a white powder(yield: 81.8%).

¹H-NMR(400 MHz, CDCl₃)δ: 1.41(3H, t, J=7.08 Hz), 1.56-1.68(2H, m),3.83-3.88(1H, m), 4.10(3H, d, J=2.20 Hz), 4.39(2H, q, J=7.08 Hz),4.85(1H, dm, J=62.99 Hz), 8.05(1H, dd, J=8.55, 10.01 Hz), 8.57(1H, d,J=1.22 Hz).

Referential Example 4

6,7-Difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid:

A mixture of ethyl 6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylate(120.8 g; 354.1 mmol), glacial acetic acid (210 ml), and conc.hydrochloric acid (420 ml) was refluxed for 6 hours, followed bycooling. The cooled reaction mixture was poured into ice/water (1500 ml)while being stirred, and the mixture was stirred for an additional 30minutes at room temperature. Crystals precipitated were collected byfiltration, and the crystals were sequentially washed with an excessiveamount of purified water, ethanol (300 ml), and diethyl ether (500 ml).The crystals were purified through recrystallization fromethanol-acetone (also through treatment with activated carbon andfiltration), followed by drying under reduced pressure at 70° C., tothereby yield 107.0 g of the title compound as white needles (yield:96.5%).

¹H-NMR(400 MHz, CDCl₃)δ: 1.64-1.75(2H, m), 3.97-4.00(1H, m), 4.17(3H, d,J=2.20 Hz), 4.91(1H, dm, J=63.23 Hz), 8.05(1H, dd, J=8.55, 10.01 Hz),8.84(1H, s), 14.31(1H, s).

Referential Example 5

6,7-Difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate:

6,7-Difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (90.30 g; 288.3 mmol) was suspended in dry diethylether (1000 ml),and boron trifluoride/diethylether complex (653 ml) was added dropwiseto the suspension under ice cooling. After completion of the addition,the reaction suspension was stirred for 24 hours at room temperature,and crystals precipitated were collected by filtration, followed bywashing with an excessive amount of dry diethylether. The washedcrystals were dried under reduced pressure at room temperature, tothereby yield 96.47 g of the title compound as a white powder (92.7%).

¹H-NMR(400 MHz, CDCl₃)δ: 1.77-1.98(2H, m), 4.30(3H, d, J=2.93 Hz),4.38-4.44(1H, m), 5.03(1H, dm, J=62.50 Hz), 8.17(1H, dd, J=8.06, 8.79Hz), 9.14(1H, s).

Referential Example 6

6,7-Difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate (alternative synthesis method):

A mixture of ethyl 6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylate(260 mg; 0.733 mmol) and tetrafluoro boric acid (42%: 5 ml) was stirredin an oil bath at 90° C. for 3 hours. The reaction mixture was cooled,and an excessive amount of purified water was added to the solution.Crystals that precipitated were collected by filtration, andsequentially washed with purified water (excess amount) and diethylether(excess amount). The crystals were collected by filtration, and driedunder reduced pressure at room temperature, to thereby yield 241 mg ofthe title compound as a white powder (yield: 91.1%). The ¹H-NMR data ofthe reaction product was in agreement with the data of that synthesizedin accordance with other synthesis methods.

Example 1

(−)-7-{(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl}-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (Compound 1a):

(7S)-7-Amino-5-azaspiro[2.4]heptane dihydrochloride (61.4 g; 0.332 mol)was dissolved in dimethylsulfoxide (800 ml), and triethylamine (138 ml;0.994 mol) was added to the resultant solution under a nitrogenatmosphere at room temperature, followed by stirring for 10 hours.6,7-Difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate (100 g; 0.276 mol) in powder form was slowlyadded to the reaction mixture, followed by stirring for 40 hours at roomtemperature. The reaction mixture was concentrated under reducedpressure, and ethanol (90%: 1000 ml) and triethylamine (20 ml) wereadded thereto, followed by refluxing for 2.5 hours. The reaction mixturewas left to cool, and crystals that precipitated were collected byfiltration. The crystals were sequentially washed with ethanol andether, followed by drying under reduced pressure at 70° C. for 16 hours,to thereby yield 72.5 g of the title compound (as 0.5 hydrate) as a paleyellow powder (yield: 61.4%). Solvents were removed from the filtrateunder reduced pressure, and water (2000 ml) was added to the residue. Anaqueous sodium hydroxide solution (3N) was added to the mixture so as toadjust the pH value to 10.0 while being stirred under ice cooling.Subsequently, an aqueous hydrochloric acid solution (3N) was added tothe mixture so as to adjust the pH to 7.4, followed by stirring for 16hours at room temperature. Crystals that precipitated were collected byfiltration, washed with water, and dried under reduced pressure at 70°C., to thereby yield 19.2 g of the title compound (as 0.5 hydrate) as apale yellow powder (yield: 33.6%).

¹H-NMR(400 MHz, 0.1N NaOD)δ: 0.53-0.59(2H, m), 0.62-0.66(1H, m),0.78-0.82(1H, m), 1.38-1.60(2H, m), 3.07(1H, s), 3.39(1H, dd, J=10.3,26.0 Hz), 3.52(3H, s), 3.72(1H, d, J=10.0 Hz), 3.89-4.00(2H, m),4.93(1H, dm, J=64.2 Hz), 7.62(1H, d, J=14.2 Hz), 8.43(1H, s).

Elementary analysis: on the basis of C₂₀H₂₁F₂N₃O₄.0.5H₂O

Calculated: C, 57.97; H, 5.35; N, 10.14. Found: C, 57.97; H, 5.31; N,10.11.

Specific rotation: [α]_(D) ²²−25.5°(c=0.832, 0.1N NaOH).

Melting point: 207-209° C.

Example 2

(−)-7-{(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl}-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid monohydrochloride monohydrate (Compound 1):

In a 3-liter teardrop flask, crystalline(−)-7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid 0.5 hydrate (61.3 g; 148 mmol: calculated based on the free form:60.0 g) was suspended in 2-propanol (720 ml). Subsequently, hydrochloricacid (5N: 59.2 ml; 296 mmol) was slowly added dropwise to the suspensionwhile being stirred under ice cooling. The thus-obtained mixture wasbrought to room temperature, and distilled water (420 ml) was addedthereto, followed by stirring for 10 minutes. The mixture was heated to60° C. in a water bath while being stirred. After the suspension turnedinto a transparent solution, activated carbon (3 g) was added thereto,and the admixture was stirred for 20 minutes at an external temperatureof 80° C. The activated carbon was filtered off, and the filtrate wasconcentrated under reduced pressure, followed by concentrated to drinessthrough evaporation. The residue was dried in a water bath at 70° C. for1 hour by use of a vacuum pump, and 2-propanol (96%: 1800 ml) was addedthereto, followed by stirring in a water bath at 80° C. After ahomogenous (clear) solution was obtained, the solution was stirred at60° C. After a while, crystals began to precipitate, when thetemperature of the water bath was brought to 25° C. over about 1.5hours, followed by slow stirring for 20 hours. Crystals thatprecipitated were collected by filtration, washed with 2-propanol, anddried under reduced pressure at 70° C., to thereby yield 56.3 g of thetitle compound as pale yellow crystals (yield: 82.7%).

¹H-NMR(400 MHz, 0.1N NaOD)δ: 0.57-0.70(3H, m), 0.81-0.85(1H, m),1.40-1.64(2H, m), 3.13(1H, t, J=4.39 Hz), 3.46(1H, dd, J=10.5, 24.6 Hz),3.60(3H, s), 3.84(1H, dd, J=7.81, 10.3 Hz), 3.99-4.06(2H, m), 5.01(1H,dm, J=64.5 Hz), 7.66(1H, d, J=14.1 Hz), 8.42(1H, d, J=1.95 Hz).

Elementary analysis: on the basis of C₂₀H₂₁F₂N₃O₄.1.0HCl.1H₂O

Calculated: C, 52.24; H, 5.26; N, 9.14. Found: C, 52.15; H, 5.25; N,9.07.

Specific rotation: [α]_(D) ²²−166.5° (c=0.990, H₂O).

Melting point: 199-208° C.

Example 3

(−)-7-{(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl}-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid-difluoroboron chelate (1b):

(7S)-7-Amino-5-azaspiro[2.4]heptane dihydrochloride (615 mg; 3.32 mmol)and triethylamine (1.40 ml) in dimethylsulfoxide (5 ml) was stirred for20 minutes at room temperature. 6,7-Difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate (1.00 g; 2.77 mmol) was added thereto, andthe resultant mixture was stirred for 20 hours at room temperature. Thereaction mixture was concentrated under reduced pressure, and purifiedwater (50 ml) was added thereto. The pH of the resultant mixture wasadjusted to 7.0 with aqueous 1N NaOH solution, and the aqueous layer ofthe mixture was taken up with chloroform (100 ml×5). The organic layerwas dried over sodium sulfate, and the solvent was evaporated. Theresidue was purified through recrystallization from ethanol, to therebyyield 1.14 g of the title compound as pale yellow crystals (yield: 91%).

¹H-NMR (400 MHz, CDCl₃)δ: 0.65-0.73(3H, m), 0.82-0.86(1H, m),1.50-1.60(1H, m), 1.66-1.76(1H, m), 3.25-3.27(1H, m), 3.45-3.58(2H, m),3.69(3H, s), 4.00-4.03(1H, m), 4.12-4.15(1H, m), 4.19-4.24(1H, m),4.95(1H, dm, J=62.7 Hz), 7.91(1H, d, J=13.7 Hz), 8.85(1H, d, J=2.20 Hz).

IR(KBr disk) cm⁻¹: 3396, 3080, 3001, 2941, 2883, 1716, 1631, 1560, 1522,1441, 1363, 1331, 1288, 1257, 1225.

Melting point: 194-197° C. (decomposed)

Elementary analysis: on the basis of C₂₀H₂₀BF₄N₃O₄.0.25H₂O

Calculated: C, 52.48; H, 4.51; N, 9.18. Found: C, 52.33; H, 4.36; N,9.01.

Specific rotation: [α]_(D) ^(19.7)=−29.3°(c=1.03, DMF)

Example 4

(−)-7-{(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl}-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (1):

(−)-7-{(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl}-6 -fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate (1.14 g; 2.52 mol) was dissolved in 80%hydrated ethanol (100 ml; prepared by mixing 4 volumes of ethanol andone volume of water). Triethylamine (2 ml) was added thereto and theresultant mixture was subjected to reflux for 3 hours. The solvent wasevaporated, and the residue was dissolved by the addition ofconcentrated HCl (5 ml) and 1N—HCl (5 ml), followed by washing withchloroform (100 ml×3). The pH of the resultant acidic solution wasadjusted to 8.0 in an ice bath with aqueous 10N NaOH solution andaqueous 1N NaOH solution, followed by stirring for three hours at roomtemperature (pH after completion of stirring=7.5). Crystals precipitatedwere collected by filtration and dried under reduced pressure, tothereby yield 980 mg of a crude form of the title compound as paleyellow crystals. The crystals were purified through recrystallizationfrom a mixture of 28% ammonia water and ethanol, then dried underreduced pressure, yielding 561 mg of the title compound as yellowishwhite crystals (yield: 55%).

¹H-NMR (400 MHz, 0.1N—NaOD) δ: 0.53-0.59(2H, m), 0.62-0.66(1H, m),0.78-0.82(1H, m), 1.38-1.60(2H, m), 3.07(1H, s), 3.39(1H, dd, J=10.3,26.0 Hz), 3.52(3H, s), 3.72(1H, d, J=10.0 Hz), 3.89-4.00(2H, m),4.93(1H, dm, J=64.2 Hz), 7.62(1H, d, J=14.2 Hz), 8.43(1H, s).

Example 5

7-[(7S)-5-Aza-7-tert-butoxycarbonylaminospiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate:

To 6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate (1.00 g; 2.77 mmol) in dimethylsulfoxide (5ml), (7S)-5-aza-7-tert-butoxycarbonylaminospiro[2.4]heptan (706 mg; 3.32mmol) and triethylamine (927 μl) were added. The resultant mixture wasstirred at room temperature for 20 hours. The reaction mixture wasconcentrated under reduced pressure, and purified water (40 ml) wasadded to the residue. Crystals precipitated were washed sequentiallywith purified water and a small amount of diethyl ether. The thus-washedcrystals were dissolved in chloroform (100 ml), and the resultantsolution was washed with water (50 ml×2) and saturated brine (50 ml).The organic layer was dried over sodium sulfate, and the solvent wasevaporated. The thus-obtained crude product was purified throughrecrystallization from a mixture of n-hexane and ethanol, followed bydrying under reduced pressure, to thereby yield 1.47 g of the titlecompound as pale yellow crystals (yield: 96%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.69-0.79(2H, m), 0.83-0.97(2H, m),1.43-1.53(1H, m), 1.45(9H, s), 1.68-1.77(1H, m), 3.49-3.52(1H, m),3.70(3H, s), 3.79(1H, d, J=11.5 Hz), 3.88(1H, s), 4.00-4.03(1H, m),4.16-4.22(1H, m), 4.23-4.25(1H, m), 4.76(1H, br.s), 4.96(1H, dm, J=62.7Hz), 7.90(1H, d, J=13.7 Hz), 8.84(1H, d, J=2.44 Hz).

IR(KBr disk)cm⁻¹: 3450, 3415, 3082, 3001, 2976, 2935, 2881, 1716, 1631,1568, 1525, 1444, 1365, 1331, 1286, 1257.

Melting point: 152-155° C.

Elementary analysis: on the basis of C₂₅H₂₈BF₄N₃O₆

Calculated: C, 54.27; H, 5.10; N, 7.59. Found: C, 54.12; H, 5.13; N,7.41.

Specific rotation: [α]D^(19.7)=−23.9° (c=1.00, CHCl₃)

Example 6

7-[(7S)-5-Aza-7-tert-butoxycarbonylaminospiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid:

7-[(7S)-5-Aza-7-tert-butoxycarbonylaminospiro-[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid difluoroboron chelate (1.47 g; 2.66 mmol) was dissolved in 80%hydrated ethanol (50 ml). Triethylamine (2 ml) was added thereto, andthe resultant mixture was subjected to reflux for three hours. Thesolvent was evaporated under reduced pressure, and aqueous 10% citricacid solution (50 ml) was added to the residue. The mixture wasextracted with chloroform (100 ml×2). The organic layer was washed withsaturated brine (50 ml), and then dried over sodium sulfate, therebyremoving the solvent. The residue was purified through recrystallizationfrom a mixture of n-hexane and chloroform, followed by drying underreduced pressure, to thereby yield 1.37 g of the title compound as paleyellow crystals (quantitative yield).

¹H-NMR (400 MHz, CDCl₃)δ: 0.64-0.75(2H, m), 0.81-0.94(2H, m), 1.45(9H,s), 1.49-1.52(1H, m), 1.54-1.62(1H, m), 3.37(1H, d, J=10.5 Hz), 3.62(3H,s), 3.63-3.67(1H, m), 3.83-3.90(3H, m), 4.06-4.10(1H, m), 4.76-4.79(1H,m), 4.85(1H, dm, J=62.7 Hz), 7.83(1H, d, J=13.5 Hz), 8.70(1H, d, J=2.20Hz).

IR(KBr disk)cm⁻¹: 3448, 3361, 3074, 2979, 2935, 2881, 1734, 1693, 1622,1512, 1448, 1367, 1325, 1352, 1252.

Melting point: 167-169° C.

Elementary analysis: on the basis of C₂₅H₂₉BF₂N₃O₆.0.5H₂O.

Calculated: C, 58.36; H, 5.88; N, 8.17.

Found: C, 58.50; H, 5.70; N, 8.17.

Specific rotation: [α]D^(19.7)=−95.2° (c=0.930, CHCl₃)

Example 7

(−)-7-{(7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl}-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (1):

7-[(7S)-5-Aza-7-tert-butoxycarbonylaminospiro-[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (1.37 g; 2.66 mmol) was dissolved by the addition of concentratedhydrochloric acid (5 ml) and 1N HCl (5 ml) on ice. The resultantsolution was washed with chloroform (100 ml×3). The pH of the resultantacidic solution was adjusted to 11.0 in an ice bath with aqueous 10NNaOH solution. Subsequently, the pH of the obtained basic solution wasadjusted to 7.4 with concentrated HCl and 1N HCl, followed by stirringfor three hours at room temperature (pH after completion ofstirring=7.4). Crystals precipitated were collected by filtration anddried under reduced pressure, to thereby yield 1.01 g of a crude form ofthe title compound as pale yellow crystals. The crystals were purifiedthrough recrystallization from a mixture of 28% ammonia water andethanol, yielding 351 mg of the title compound as yellowish whitecrystals (yield: 33%).

¹H-NMR(400 MHz, 0.1N—NaOD)δ: 0.53-0.59(2H, m), 0.62-0.66(1H, m),0.78-0.82(1H, m), 1.38-1.60(2H, m), 3.07(1H, s), 3.39(1H, dd, J=10.3,26.0 Hz), 3.52(3H, s), 3.72(1H, d, J=10.0 Hz), 3.89-4.00(2H, m),4.93(1H, dm, J=64.2 Hz), 7.62(1H, d, J=14.2 Hz), 8.43(1H, s).

Referential Example 7

Methanesulfonic Acid Salt of(−)-7-{(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl}-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid:

(−)-7-((7S)-7-Amino-5-azaspiro[2.4]heptan-5-yl)-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylicacid (1a) (2.51 g) was suspended in ethanol (20 ml). Methanesulfonicacid (1.2 equivalents) was added thereto, and the resultant mixture wasstirred for five minutes at room temperature. Subsequently, diethylether (80 ml) was added thereto. Crystals precipitated were washed withdiethyl ether, and collected through filtration (2.01 g, 94%).

The above-obtained crude crystals of methanesulfonic acid salt (900 mg)was dissolved in hot isopropanol (100 ml), and the solution wasconcentrated under heating until the volume of the entire solutionbecame 40 ml. The concentrate was allowed to cool at room temperature,and crystals precipitated were collected through filtration, followed bywashing with isopropanol, thereby yielding 720 mg of the title compound(yield: 80%).

Melting point: 257-258° C.

¹H-NMR(400 MHz, 0.1N—NaOD)δ: 0.58-0.72(3H, m), 0.80-0.90(1H, m),1.40-1.62(2H, m), 2.82(3H, s), 3.10-3.12(1H, m), 3.41-3.49(2H, m),3.58(3H, s), 3.81(1H, dd, J=2.44, 9.77 Hz), 4.85-4.93, 5.04-5.07(each0.5H, m), 7.65(1H, d, J=14.16 Hz), 8.42(1H, s).

Test Example 1 (Confirmation of the Crystal Morphology of Compound (1))

-   (1) FIG. 1 shows the results of powder X-ray diffractometry of    compound (1) (by means of an X'pert powder X-ray diffraction    apparatus, product of Philips), and FIG. 2 shows the IR spectrum of    compound (1) (obtained using model FT-720, FT-IR, product of    HORIBA). Thermal analysis of compound (1) revealed that the weight    loss was 4.2% by weight, which was in agreement with the theoretical    value (3.9%) of monohydrate.-   (2) Quantitative analysis by the Karl Fischer's method revealed that    the water content of compound (1) is 4.11%, which is in agreement    with the result of thermal analysis.

Test Example 2

Moisture absorption/desorption of compound (1) was investigated by usingsamples each weighing approximately 10 mg and a moisture absorptionanalyzer manufactured by VTI (model SGA-100). The measurement wasperformed at 25° C., and the relative humidity was changed in the rangeof 5% to 95% at intervals of 5% or 10%. When the weight change of asample is 0.03% or less within 30 minutes, the sample is considered tobe in a state of equilibrium, and in this test, the longest equilibriumtime was set to be 180 minutes. The weight change of each sample atdifferent relative humidities was determined. As a result, as shown inFIG. 3, compound (1) was found to be stable, neither absorbing ordesorbing moisture within the relative humidity range of 5-95% RH.

Test Example 3

Acid addition salts of compound (1a) (methanesulfonic acid salt,p-toluenesulfonic acid salts, citric acid salt, and malic acid salt)were prepared, and their stability under various levels of humidity wasevaluated. All the tested salts showed a tendency toward moistureabsorption under high-humidity conditions. In the meantime, althoughattempts were made to prepare an acetic acid salt and a lactic acid saltof compound (1a), no salt was formed.

Test Example 4

Solubility of compound (1) in water was investigated. Compound (1) wasfound to have a high solubility in water; i.e., 100 mg/mL or more.

Test Example 5

Compound (1) (1.5 mg) was stored for one week under the followingconditions: (1) in a sealed bottle at 70° C., (2) in an open-air stateat 50° C., 75% RH (NaCl), or (3) irradiation with light at 100,000 lx·h(2,500 lx×40 h). Subsequently, the residual amount of compound (1) wasquantitatively determined by means of liquid chromatography. Results areas follows: Compound (1) remained stable, not undergoing anydecomposition, even when exposed to light irradiation. Moreover, thestability of compound (1) when exposed to light was superior to that ofcompound No. 26bb disclosed in Japanese Patent No. 2714597.

Test Example 6

Slc:ddY mice (male; 3 weeks old) were used in groups of ten. Compound(1) was dissolved in distilled water in order to prepare injections, andthe resultant solution was cisternally administered in a volume of 5μg/mouse. To the members of the group where compound (1) andbiphenylacetic acid were used in combination, biphenylacetic acid (400mg/kg) was perorally administered first, and 30 minutes following theadministration, compound (1) was cisternally administered in a mannersimilar to that employed for the single use of compound (1).

The results are as follows: In both cases of solo administration ofcompound (1) and combined administration of compound (1) andbiphenylacetic acid, compound (1) caused neither spasm nor death and wasproven to have very weak central toxicity. Thus, compound (1) is a verysafe compound.

In contrast, administration of comparative compound A at the same doseinduced spasm in two mice out of ten. Also, there was one death amongthe ten mice. Moreover, in the case of combined administration withbiphenylacetic acid as described above, there were four spasm cases andtwo deaths out of 10 mice.

Comparative Compound A:

Test Example 7

Groups of young beagles (male; 3-4 months old), each group consisting of3 dogs, were used in the test. Compound (1) was perorally administeredto each dog for eight days. Thereafter, important diarthroses werepathologically examined. Results: Members of the groups to whichcompound No. 26bb disclosed in Japanese Patent No. 2714597 wasadministered at high doses of 14.1 mg/kg or more showed formation ofblebs or erosions in arthrodial cartilage, whereas in the compound (1)administration groups (7.5 mg/kg, 15 mg/kg, and 30 mg/kg), formation ofblebs or erosions was not at all observed. Thus, articular toxicity ofcompound (1) is quite weak and insignificant, proving that compound (1)is a very safe compound.

Test Example 8

Groups of Balb/c mice (female; 5 weeks old), each group consisting of5-6 mice, were used in the test. After compound (1) was intravenouslyadministered to each mouse, the mouse was exposed to long-wavelength UVlight (UV-A) (20 J/cm²) for four hours. Thereafter, the auricle wasvisually observed for 96 hours. Subsequently the mouse was sacrificedfor examination of the tissue. In the groups where compound (1) wasadministered at (100 mg/kg), no abnormality was observed either duringvisual observation or at the time of tissue examination. Thus, compound(1) is a very safe compound, being free from phototoxicity, which isoften observed with quinolone-based antibacterial agents.

Test Example 9

(1) Therapeutic Effect on Mouse Pneumonia Model by Use of Pneumococcusof Low Penicillin Sensitivity:

Groups of CBA/J mice, each group consisting of 5 mice, were used in thetest. The mice were nasally infected with pneumococcus SPI-13 at 5.3×10⁶CFU/mouse. Compound (1) was subcutaneously administered to each mouse atdoses of 7.5 mg/kg, 15 mg/kg, or 30 mg/kg, for three consecutive daysfrom the day following infection, twice a day at an interval of 6 hours.The efficacy of compound (1) was assessed by counting the number of theintrapulmonary bacteria on the day following the final administration.Results: At a dose of 30 mg/kg or 15 mg/kg, compound (1) reduced thebacterial count to below the detection limit, and at a dose of 7.5mg/kg, reduced the bacterial count to approximately half that of thecontrol.

(2) Infection-preventing Effect on Mouse Sepsis Model.

Groups of Slc:ddY mice, each group consisting of 7 mice, were used inthe test. The mice were intraperitoneally inoculated withmethicillin-resistant Staphylococcus aureus (MRSA) strain 7866 (1.07×10⁸CFU/mouse) or E. coli strain E77156 (8.08×10⁷ CFU/mouse). Compound (1)was given as a single injection into the tail vein of each mouseimmediately after infection. On the basis of the survival count on day 7after infection, 50% efficacy was calculated using the probit method, tothereby evaluate the efficacy. Results: The 50% efficacy of compound (1)against MRSA strain 7866 was found to be 3.34 mg/kg, and the sameefficacy against E. coli strain E77156 was found to be 0.57 mg/kg.

From (1) and (2) above, it can be concluded that compound (1) hasexcellent preventive and therapeutic effects against differentinfectious diseases when tested in vivo.

Test Example 10 (Antibacterial Activity)

Antibacterial activity was investigated by comparing compound (1) withcomparative compounds B and C (see next page). This test was performedaccording to the standard method recommended by Japanese Society ofChemotherapy. The test results are shown in Table 1.

TABLE 1 Antibacterial Activity MIC(μg/mL) Comparative ComparativeCompound (1) Compound B Compound C E. coli, NIHJ ≦0.003 0.006 0.025 Pr.vulgaris, 08601 0.012 0.05 0.05 Ser. marcescens, 10100 0.05 0.20 0.20Ps. aeruginosa, 32104 0.20 0.39 0.78 Ps. aeruginosa, 32121 0.05 0.200.39 S. aureus, 209P 0.006 0.025 0.05 S. epidermidis, 56500 0.05 0.100.20 Str. faecalis, ATCC 19433 0.10 0.20 0.39

Comparative Compound B

Comparative Compound C

The compounds of the present invention are endowed with excellentantibacterial activity and safety, and also are very stable againstlight or humidity, thereby finding utility as antibacterial agents.

1. A compound represented by the following formula (1)


2. A compound as claimed in claim 1, which assures crystals exhibitingcharacteristic peaks in the vicinity of angles of diffraction (2θ) of6.9, 10.5, 14.4, 23.1, 26.9, and 27.8(°) when subjected to powder X-raydiffractometry.
 3. A solid antibacterial composition containing acompound represented by the following formula (1) together with apharmaceutically acceptable carrier


4. A solid antibacterial composition as claimed in claim 3, wherein thecompound of formula (1) assumes crystals exhibiting characteristic peaksin the vicinity of angles of diffraction (2 θ) of 6.9, 10.5, 14.4, 23.1,26.9, and 27.8(°) when subjected to powder X-ray diffractometry.
 5. Amethod for the treatment of bacterial infection in a mammal or fish,which comprises administering to said mammal or fish an effective amountof a compound represented by the following formula (1)


6. The method as claimed in claim 5, wherein the compound of formula (1)assumes crystals exhibiting characteristic peaks in the vicinity ofangles of diffraction (2 θ) of 6.9, 10.5, 14.4, 23.1, 26.9, and 27.8(°)when subjected to powder X-ray diffractometry.